First trimester extravillous trophoblast secretes HLA class I molecules via small extracellular vesicles

Human pregnancy requires acceptance and support for the semi-allogeneic embryo and effective protection of both mother and fetus. A failure to adapt, from either side, may cause abortion. The placenta-derived extracellular vesicles (EVs) have a crucial role in human implantation and pregnancy. These are lipid bilayer membrane-delimited, nano-to-micro sized extracellular microvesicles of endosomal origin, containing diverse signaling molecules, and functioning as short and long-distance messengers. We have already shown that first-trimester placenta releases the soluble HLA-C and HLA-G KIR ligands to modulate maternal cytotoxicity via the KIR/HLA axis. This study is to find whether extravillous trophoblast (EVT) secretes these HLA class I molecules via small EVs.

sEVs were isolated by ultrafiltration or precipitation from serum-free conditioned media from primary trophoblast-derived EVT, and non-tumor EVT-like model Sw71 cell line, cultured as monolayer and spheroids. sEVs from cultured placental explants served as a positive control. Combined data from several methods was used for their characterization including BCA, DLS, TEM, IEM, Dot blot, and FACS.

Primary trophoblast-derived EVT and Sw71 EVT-like cells produced intact and well-visible CD63⁺, HLA-G- and HLA-C-bearing sEVs, regardless of culture mode and type of isolation. Both methods yielded sEVs sized 30-100 nm.

We show original data on the HLA-C secretion via sEVs by early pregnancy EVT and confirm the production of HLA-G-positive sEVs. A new asset to the usefulness of the Sw71 spheroid model as an implanting blastocyst surrogate is added as a tool to elucidate the sEV-based signalization in the implantation.